The invention relates to an arrangement for the detection of biochemical or chemical substances using fluorescent light excitation, and to a process for the production of such an arrangement. The arrangement according to the invention can be used in a variety of fields, for example in biotechnology, in molecular medicine, in pharmaceuticals development and for the analysis of various chemical substances.
For a considerable time, a wide variety of optical methods and systems have been used, for example for research into various biological systems and processes, in microbiology and molecular medicine. In this case, spectrometers are often used, which permit a relatively high information content with correspondingly high accuracy when they are employed. Spectrometers are, however, unsuitable for many routine experiments that are encountered, since elaborate preparation of the individual samples is required, and the time needed for the measurements is too long and can lead to denaturing of the samples.
For many applications, solutions are required with which rapid, accurate and inexpensive analysis of a very large number of samples (of the order of 106) can be carried out. Examples of such applications include DNA libraries.
A variety of measurement techniques have to date been used for analysis and detection. For example, variation in absorption, variation in refractive index or the fluorescent light excited are determined. In the case of measuring the intensity of fluorescent light, the excitation light and the modified fluorescent light can be directed parallel or perpendicular to one another.
If the two different types of light are directed perpendicular to one another, the formation of an evanescent field along a waveguide is utilized. This form is used, for example, to detect antigen-antibody reactions. In these so-called xe2x80x9csolid-phase fluoroimmunoassaysxe2x80x9d, the detection-specific antibodies are immobilized on a sensor surface. The analyte (antigen) is bound to a corresponding antibody and can then be detected by being made to fluoresce by the evanescent field either directly or with the use of a marker. Excitation of fluorescence by the evanescent field of a waveguide gives the advantage that the penetration depth of the evanescent field is limited (about 100 to 200 nm) and only analytes or markers bound directly to the sensor surface are therefore excited. The result of this is that the measurable intensity of the fluorescent light is a direct measure of the number or concentration of bound analyte or marker, and for this reason it is possible to do without additional washes to remove unbound marker molecules.
Both optical fibres and layer waveguides can be used for such optical waveguides. Optical fibres have the advantage that corresponding sensors or devices are simple and inexpensive to produce. They can be used in virtually any places, even ones which are very inaccessible, and the measurement signals can be transmitted optically with ease over considerable distances. For example, U.S. Pat. Nos. 4,447,546 and 4,909,990 disclose that, although optical fibres can in principle be used for corresponding processes, flat layer waveguides are generally used.
Flat layer waveguides have the advantage that a very wide variety of substances that been to be analysed, and immobilizing layers which may be necessary, can be applied and structured with ease. Examples of methods which can be used for this include spin-on coating, pouring, sputtering and known vacuum evaporation methods. Furthermore, a plurality of individual sensors can be produced on a single large plate, with the sensors produced in this way having virtually the same properties. Another advantage of such sensor structures is that they are very stable, and consequently are also very easy to handle. A very wide variety of layer materials can be applied and structured with ease. In this case, a very wide variety of metals, glasses and polymers can be used.
It is readily possible for flat laminar structures to be used in detection instruments, and for optical measurement methods to be carried out in them.
The production and structuring of flat structures of this type has been tried and tested from previous experience in microelectronics fabrication, and the associated costs are therefore relatively low.
Biological sensor structures which use the formation of evanescent fields for fluorescent light excitation are, for example, [lacuna] by S. Sjxc3x6lander and C. Urbaniczky: Integrated Fluid Handling System for Biomolecular Analysis, Anal. Chem., 63 81991) 2338-2345 and R. Cush et al.: The resonant mirror: a novel optic biosensor for direct sensing of biomolecular interactions. Part 1: principles of operation and associated instrumentation. Biosensors Bioelectron., 8 (1993) 347-353 and J. E. Fletcher et al.: A Rapid, Biosensor-based, assay for PSA in Whole Blood, Tumor Marker Update Vol. 5, No. 5 (1993). The detection limits for the sensor described by S. Sjxc3x6lander and C. Urbaniczky are thus 0.5 ng/ml (FCFD).
Another device for fluorescent detection of biological reactions using evanescent field excitation of a layer waveguide is described in WO 94/27137, in which case there is a detection limit when using a reference channel at 10xe2x88x9213 molar solutions.
In this solution, use is made of a planar waveguide on whose surface separate fields are provided, on or in which trapping molecules are immobilized and it is also possible for different samples to be determined using evanescent field excitation in the form of fluorescence immunoassays for the different fields. In this case, the light is necessarily injected into the planar waveguide through an end face of the planar waveguide, and in a preferred embodiment through a lens-like configuration of this end face, and the evanescent field is formed and the fluorescence excited at the interface. The fluorescent light emerges at the opposite side of the waveguide and can be measured using the usual detectors, it being possible to control the optical path using various optical elements. This gives rise to two significant disadvantages, which, as mentioned above, have a negative effect on the measuring sensitivity. On the one hand, it is not possible to prevent the different samples that the fields contain from affecting the fluorescent light, since complete optical isolation is impossible, and on the other hand problems arise owing to the injection of the light exclusively through the end of the waveguide, so that it is necessary to put up with relatively large losses of light.
Although, in this case, the use of appropriate optical filters has been proposed to reduce the effect of stray light, these also cause loss of fluorescent light and the aforementioned effect of the present light from the various samples cannot be prevented entirely.
EP 0 519 622 A2 describes another device for carrying out assays using evanescent field excitation, in which tapering openings in a silicon substrate and the containment thus obtained is filled with a liquid by which an ion-selective membrane is formed.
The solutions described by S. Sjxc3x6lander and C. Urbaniczky, and R. Cush et al., detect changes in refractive index that are caused by the build-up of analytes on the surface. The measured change in refractive index does not, however, involve only the particular build-up to be investigated, and the selectivity that can be obtained is consequently not always what it could be. For the measurements carried out in that way, very high outlay on equipment is necessary, which is also reflected in relatively high costs. It is not possible to examine a plurality of samples at the same time.
In the biosensor described by J. E. Fletcher et al., the relatively extensive excitation of fluorescence, and its evanescent injection into a layer waveguide in FCFD, leads to relatively low sensitivity.
EP 0 723 146 A1 and EP 0 194 132 A2 describe other possible ways of detecting biochemical or chemical substances using fluorescent light excitation, both cases describing the arrangement of different samples in a predetermined system and their evaluation. EP 0 723 146 A1 describes examples in which various samples are immobilized next to one another, and CCDs or individual lenses in an arrangement designed according to the sample arrangement are used for the evaluation.
The object of the invention is to provide a way in which it is possible to detect a relatively large number of samples relatively simply and with high accuracy in a very short time.
This object preferably is achieved by the characterizing features of the present invention. Advantageous embodiments and further developments of the solution will be apparent from the description of the invention provided herein.
The arrangement according to the invention consists essentially however, it is also possible to use a lens matched to the structuring, for example a Fresnel lens, for this. A buffer layer, and a waveguide array formed corresponding to the structuring made in the substrate, can be applied on the other side of the substrate from the lens array.
Each of the microstructures made in the substrate defines a fluorescence excitation and detection channel respectively for one of the samples to be detected.
There are principle two ways of exciting the fluorescent light. First, the evanescent field may be formed in a waveguide array that is formed on the side of the substrate opposite the detector side. The second possible excitation method is to direct the excitation light on to the individual samples through the lens array matched to the structuring in the substrate, it being in each case possible for the fluorescent light to be focused on to the detector using the lens array arranged on the detector side of the substrate. For this purpose, the detector is likewise designed in the form of an array, for example as a CCD array, so that the fluorescent light from each individual sample can be detected separately.
For the case of evanescent field excitation, however, another possibility is to do without a lens array and, instead, to fit a detector array matched to the structuring on the substrate, or arrange the latter in such a way that the various samples can be detected selectively.
The structuring of the substrates, which may for example have the 4xe2x80x3xc3x974xe2x80x3 format known from a semiconductor technology, is easy and inexpensive to produce using the silicon processing methods known from this field, and in this case, microstructuring can be achieved on such a substrate which allows a sample number of up to 106/cm2.
The substrate to be used according to the invention can advantageously ensure that a very wide variety of fluorescent signals, which are to be evaluated using the detector array, do not affect one another or become superimposed, so that each sample can be uniquely assigned a measurement without interference. To that end, the substrate preferably consists of a material which absorbs the fluorescent light at its wavelengthis). Another way of achieving the same effect is, however, to design the respective walls in the structuring of the substrate such that they reflect, so that this undesirable effect can be avoided.
The preferred structuring in the substrate, by forming pyramid or conical, or upright openings, has the further advantage that the fluorescent light is favourably reflected and therefore concentrated in the direction of the lens array, and consequently can also be directed at the respective detectors.
With the arrangement according to the invention, it is possible to detect molecule concentrations equal to 10xe2x88x9212 molar or less, in a large number of different samples in a very short time. Further, the exciting light can be directed at the respective sample in an exactly defined way, and the respectively corresponding fluorescent light can be directed, while substantially avoiding contributions from the stray light or other interference, directly at the respective detector assigned to an individual sample, and the corresponding analyte may where appropriate also be determined quantitatively.
With the solution according to the invention, not only can improved spatially resolved measurement be carried out, but there is also optionally the possibility of taking time-resolved measurements using a corresponding time-controllable detector arrangement (e.g. triggerable CCD camera and electronic delay unit).